Fig 8.

A. Cell lysates were subjected to immunoblotting using an antibody which recognises both the Native and LTR-IL-33 protein isoforms. The predicted molecular weight of LTR-IL-33 is approximately 5kDa less than the native protein. Actin was also assessed as a loading control. B: HEK293T cells were transfected to exogenously express Flag-tagged Native IL-33, LTR-IL-33 or a series of mutants of the LTR-form where two of the three potential translation-initiating Methionines (M) were mutated to Isoleucine (I). The triple III mutant and empty vector control (EV) were also included as negative controls. A GFP expression plasmid was also co-expressed as a transfection control. Total IL-33, GFP and Actin levels were assessed by immunoblotting. C: LS513 or HUVEC cells were cultured to confluence, then lysed and the nuclear and cytosol fractions separated. Lysates were subjected to immunoblotting for IL-33, Lamin A (nuclear marker) and α/β-tubulin as a loading control.